DNA vaccine has shown considerable promise in combating a wide range of devastating diseases including cancer and bacterial and viral infections. Compared with conventional pathogen-based and subunit vaccines, DNA vaccine possesses several advantages, including the potential of eliciting both antibody and cell-mediated immune responses. Various forms of DNA vaccine are currently being evaluated in animal models and human trials. One major obstacle to successful clinical use of DNA vaccines is the difficulty of delivering DNA molecules to antigen-presenting cells (APCs) that mobilize the immune system. While viruses are widely investigated for DNA vaccine delivery application, synthetic polymers present a promising alternative avenue that is safe, inexpensive, and versatile. The long-term goal of this proposal is to develop a platform DNA vaccine delivery technology based on rationally designed polymer-based nanostructures, for safe, clinical use in humans with high potency. We hypothesize that DNA vaccine incorporated in multifunctional structurally defined polymer nanoparticles that target APCs, achieve high levels of antigen expression and presentation, and activate resting APCs, will be able to generate potent long-lasting antigen-specific immune responses in vivo. To test this hypothesis, we propose the following specific aims: (1) we will synthesize biodegradable multifunctional polymer nanoparticles and (2) evaluate their efficiency of cell targeting, gene transfection, and immune activation, using cultured cells and an in vivo mouse model. (3) As a proof-of-principle, we will immunize mice with nanoparticles carrying a model DNA vaccine, quantify antigen-specific immune responses, and determine if the mice are able to reject tumor grafts expressing the model antigen. This exploratory/developmental (R21) project is intended to establish the feasibility of this approach, setting the stage for testing vaccines against human disease antigens. With further development, the proposed nanoparticle technology is expected to have a significant impact on gene-based immunotherapy for treating cancer and infectious diseases. [unreadable] [unreadable] [unreadable]